Methods for isolating biomolecules from fixed biological specimens embedded in paraffin are known from the prior art. Within this context a fixed specimen is to be understood as a biological specimen which is made sustainable in a way known in its own right by treating, for example, with a formaldehyde solution, anhydrous ethanol or acid alcoholic solutions. In order to further improve storage stability these specimens are then transferred into paraffin. The specimens treated with formalin and embedded in paraffin are also called FFPE (“formalin-fixed, paraffin-embedded”) material. The specimens fixed and embedded using these methods can be used, for example, for histopathological examinations and/or then be kept over a very long period without any appreciable change taking place to the biomolecules contained within these specimens. In particular, even after long periods of time, the nucleic acids, i.e. RNA and DNA, can still be extracted from these specimens.
However, the extraction of these biomolecules from this type of fixed specimen embedded in paraffin is complex because the specimens must first of all be released from the paraffin surrounding them. The specimens are generally thin sections the paraffin portion of which generally considerably exceeds the specimen portion. Since the paraffin can interfere with or totally prevent further specimen preparation and isolation of the biomolecules, various methods are proposed in the prior art by means of which the paraffin can be separated out.
In WO 2006/039563 it is proposed to pre-treat the biological sample after cutting, for example with a microtome, with xylene, toluene, isoparaffin or limonene in order to dissipate the paraffin. This solution is then centrifuged, the solvent is removed, and the specimen optionally washed with ethanol in order to remove any solvent residues which may be present. Next the specimen is dried and then subjected to further analysis.
The multiple steps of washing with ethanol required in order to extract clean specimen material are on the one hand complex and, moreover, conceal the risk of parts of the specimen material inadvertently already being removed after centrifuging out when subtracting the ethanol supernatant. The same problem arises during the centrifugation and removal of the solvent for the paraffin. This is particularly problematical if only a small amount of specimen material is available because then the specimen material can hardly be identified by the naked eye in the solvent. Since the preparation of clean specimens using this method requires a great deal of skill, and above all experience, reliable automation is hardly possible.
In US 2007/0026432 a further method for the analysis of fixed biological specimens embedded in paraffin is described. For this purpose it is proposed to heat the specimen material embedded in paraffin to above the fusion point of the paraffin after adding detergents, and then to allow the specimen to cool down again to below the fusion point of the paraffin. Extraction of the specimen material is then implemented with the aid of a cannula which is moved through the solidified paraffin layer into the specimen material located beneath the latter. It is a disadvantage with this method that when penetrating the paraffin layer the cannula can become blocked with paraffin, and this makes automation practically impossible.